Cambios histopatológicos de la mucosa bucal de ratas expuestas al humo de cigarrillos / Histopathological oral tissues changes of rats exposed to tobacco smoke

Introducción: El propósito de esta investigación fue evaluar el efecto del humo del tabaco en la mucosa bucal"in vivo" usando un modelo animal. Materiales y métodos: La población de estudio estuvo constituida por 20 ratas hembras Sprague Dawley, 10 expuestas al humo de 10 cigarrillos diarios por 16 semanas (GEC) y un grupo control no expuesto (GC). Las ratas fueron sacrificadas y se extrajo la lengua para su estudio histopatológico. Variables categóricas fueron comparadas mediante la prueba U de Mann-Whitney. Valores p<0,05 fueron considerados estadísticamente significativos. Resultados: La mucosa lingual del GEC mostró mayores cambios epiteliales proliferativos que el GC, como hiperplasia basal (p= 0,001), acantosis (p= 0,0001), hipercromatismo y picnosis nuclear (p= 0,010 y 0,014 respectivamente), y displasia epitelial (p= 0,0001). En el corion de la mucosa del GEC se observó un incremento del infiltrado inflamatorio crónico (p= 0,007); y aunque no hubo aumento en el número de vasos sanguíneos en el GEC, es importante resaltar que la ubicación de los mismos fue muy próxima al epitelio de revestimiento expuesto al tabaco. Discusión: El presente estudio, usando un modelo animal, demuestra que el humo del tabaco induce cambios precoces en la arquitectura tisular y morfología de células epiteliales de la mucosa bucal de aspecto macroscópicamente sano (AU)

Introduction: The purpose of this study was to evaluate the histopathological changes in oral tissues of rats exposed to tobacco smoke. Materials & methods: 20 female rats Sprague Dawley were included in the study and 10 rats exposed to environmental tobacco smoke of 10 cigarettes daily for 16 weeks. All animals were sacrificed and tissues extracted, died using H&E and finally evaluated under light microscope. Categorical variables were compared using U Mann-Whitney test. P values<0.05 were considered statistically significant. Results: Oral mucosa of rats exposed to tobacco showed augmented epithelial changes as basal hyperplasia basal (p= 0,001), acanthosis (p= 0,0001), nuclear hyperchromatism and picnosis (p= 0,010 y 0,014 respectively), and epithelial dysplasia (p= 0.0001). Increased chronic inflammatory infiltrate was also observed in this group (p= 0.007), as well as, a higher number of blood vessels although difference was not statistically significant. Discussion: Tobacco inhalation showed to produce tissue alterations of macroscopically healthy mucosa of rats. This study, using an animal model, demonstrated microscopical oral mucosa changes produced by tobacco smoke without clinical alterations (AU)

Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs.

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.

Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14 alpha demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > or = 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.

Cure of short- and long-term experimental Chagas' disease using D0870.

Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.

Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of delta 24(25) sterol methyl transferase (E.C. 2.1.1.43) of yeasts and fungi on epimastigotes and amastigotes on Trypanosoma (Schizotrypanum) cruzi, the causative agents of Chagas disease, as well as its chemotherapeutic effects in a murine model of the disease. On the epimastigote form proliferating in liver infusion tryptose medium at 28 degrees C 22,26-azasterol (AZA), a cholestanol analog with a 6-membered aza ring as a side chain produced a dose-dependent reduction of the growth rate up to 3 microM, but at 10 microM complete growth arest and cell lysis took place after 120-144 h. For 24(R,S),25-epiminolanosterol (EIL), complete growth arrest and lysis took place with 6 microM. In both cases the antiproliferative effects were potentiated by the simultaneous incubation of the epimastigotes with inhibitors of sterol C-14 alpha-demethylase such as ketoconazole or SDZ 89,485, as indicated by concave isobolograms and fractional inhibitory concentrations ranging from 0.11 to 0.46. Analysis of the sterol composition in control and treated cells by thin-layer and capillary gas-liquid chromatography coupled to mass spectrometry showed that growth inhibition correlated with the complete disappearance of the native endogenous sterols of the parasite (ergosterol and 24-ethyl analogs) and the accumulation of 24-desalkyl sterols. Against the clinically relevant amastigote form proliferating inside cultured Vero cells at 37 degrees C, AZA eradicated the parasite of 100 nM, while the corresponding concentration for EIL was 300 nM. Synergic effects of both inhibitors when combined with ketoconazole against this form of the parasite was demonstrated using a three-dimensional analytic method which allowed the identification of optimal drug concentrations. Finally, it was found that daily oral administration of AZA at 50 mg/kg/day for a total of 43 doses to mice infected with a lethal inoculum of T. cruzi allowed survival of all treated animals 25 days after infection, while all control (untreated) animals were dead at this point of time. Increased survival correlated with a 90% reduction in parasitemia in the treated animals. The antiparasitic effects of the azasterol were potentiated in combined treatments with ketoconazole. This is the first report of a successful application of a sterol methyl transferase inhibitor as a chemotherapeutic agent in a protozoal infection.

Experimental chemotherapy with combinations of ergosterol biosynthesis inhibitors in murine models of Chagas' disease.

We report the effects of ketoconazole and the bistriazole ICI 195,739 acting alone or in combination with the allylamine terbinafine (Lamisil) on murine models of Chagas' disease. Mice infected with 10(5) Trypanosoma (Schizotrypanum) cruzi blood trypomastigotes and treated orally with 30 mg of ketoconazole per kg of body weight per day for 7 days, starting at 24 h postinoculation, had 100% survival after 35 days, while controls (untreated) or animals that received 15 mg of ketoconazole or 100 mg of terbinafine per kg/day by the same route had 0% survival after the same period of time. However, all mice receiving the combination of 15 mg of ketoconazole plus 100 mg of terbinafine per kg/day survived for 35 days after infection; it was shown that the survival of the animals treated with this combination was statistically greater than that obtained with either drug acting alone and was indistinguishable from that observed with the high doses of ketoconazole, indicating a synergistic action of the drugs in vivo. However, most animals that survived after the 7-day treatments were not cured, as indicated by a delayed but persistent parasitemia. When the treatment was extended to 14 days, 100% survival was obtained 10 weeks after inoculation for mice treated with 30 mg of ketoconazole per kg/day and the combination of 15 mg of ketoconazole per kg/day plus 100 mg of terbinafine per kg/day, while two-thirds of the mice treated with 15 mg of ketoconazole per kg/day alone were alive after the 14-day treatment; controls or animals that received 100 mg of terbinafine per kg/day did not survive after 25 days. Parasitemia in all surviving mice was negative after 55 days but parasitological cure, as assessed by subinoculation of organs in naive animals, was predominant only in animals that received the combined drug treatment. We also investigated the bistriazole ICI 195,739 and found, as reported previously, that just 1 mg of the compound per kg/day administered orally for 5 days was enough to protect most mice from death 30 days after inoculation, but no parasitological cures were observed. However, in the protocol used in the present study, the protective activity of ICI 195,739 at suboptimal doses (0.5 mg/kg/day) could be enhanced when it was used in combination with terbinafine at doses of the allylamine that by themselves induced no significant protection. Survival of the mice was inversely correlated with the levels of parasitemia in all cases. Extension of the treatment period with the triazole to 15 days at 1 mg/kg/day afforded definitive protection against death, with parasitological cure being achieved in 50% of mice at 10 weeks postinoculation, but no enhancement of its activity at suboptimal doses was observed when it was used in combination with terbinafine during this extended observation period. Taken together, these results supports the proposition that ketoconazole used in combination with terbinafine could be useful in the treatment of humans with Chagas' disease because it can promote parasitological cure without the need to resort to the use of high levels of the azole, which is known to interfere with hepatic function and steroid synthesis in the host. They also support the conclusions of previous in vitro studies which suggested that the triazole ICI 195,739 blocks the proliferation of T. cruzi by a mechanism which differs from those of classical ergosterol biosynthesis inhibitors.

Mevinolin (lovastatin) potentiates the antiproliferative effects of ketoconazole and terbinafine against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies.

We have studied the antiproliferative effects of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, on the protozoan parasite Trypanosoma (Schizotrypanum) cruzi and its ability to potentiate the action of specific ergosterol biosynthesis inhibitors, such as ketoconazole and terbinafine, both in vitro and in vivo. Against the epimastigote form in vitro, mevinolin produced a dose-dependent reduction of the growth rate up to 25 microM, but at 50 and 75 microM, complete growth arrest and cell lysis took place after 144 and 96 h, respectively. A systematic study of the effects of mevinolin combined with ketoconazole and terbinafine, which act at different points in the ergosterol biosynthesis pathway, on the proliferation of epimastigotes indicated a synergic action, as shown by concave isobolograms and fractional inhibitory concentration indexes ranging from 0.17 to 0.54. Analysis of the sterol composition and de novo sterol synthesis in control and treated cells by thin-layer and gas-liquid chromatographies showed that the antiproliferative effects of the drug alone and in combination were correlated with the depletion of the endogenous ergosterol pool and particularly with a critical (exogenous) cholesterol/endogenous 4-desmethyl sterol ratio in the cells. When we studied the effects of mevinolin on the amastigote form proliferating inside Vero cells in vitro, only very modest effects on the parasites were observed up to 0.75 microM; above this concentration, significant deleterious effects on the host cells were found. However, when the same concentration of the drug was combined with ketoconazole, it was able to reduce by a factor of 10 the concentration of the azole required to eradicate the parasite (from 10 to 1 nM), again indicating a synergic action. On the other hand, a combination of mevinolin and terbinafine had only additive effects on amastigotes, but a ternary combination of mevinolin, ketoconazole, and terbinafine was again clearly synergistic. In vivo studies with a murine model of Chagas' disease showed that mevinolin can also potentiate the therapeutic effects of ketoconazole in this system; combined treatment with the two drugs at doses that alone offered only limited protection against the parasite was able to essentially eliminate circulating parasites and produce complete protection against death. These results confirm the synergic action against the proliferative stages of T. cruzi both in vitro and in vivo and in vivo of combined ergosterol biosynthesis inhibitors that act at different points in the pathway and suggest that mevinolin combined with azoles, such as ketoconazole, can be used in the treatment of human Chagas' disease.

Experimental heteroxenous cycle of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae) in white mice and in cats.

Reports of natural infections of sylvatic carnivores by adult worms of species similar to Lagochilascaris minor in the Neotropical region led to attempts to establish experimental cycles in laboratory mice and in cats. Also, larval development was seen in the skeletal muscle of an agouti (Dasyprocta leporina) infected per os with incubated eggs of the parasite obtained from a human case. In cats, adult worms develop and fertile eggs are expelled in the feces; in mice, larval stages of the parasite develop, and are encapsulate in the skeletal muscle, and in the adipose and subcutaneous connective tissue. From our observations, we conclude that the larva infective for the mouse is the early 3rd stage, while for the final host the infective form is the later 3rd stage. A single moult was seen in the mouse, giving rise to a small population of 4th stage larvae, long after the initial infection.

The heterogeneity of Leishmania cell-surface antigens.

A comparative study of the radioiodinated promastigote cell-surface antigens of Leishmania mexicana and L. major was carried out under reduced and nonreduced conditions by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Under reduced conditions, the cell surface of L. mexicana promastigotes showed three iodinated polypeptides with molecular weights of 65,000, 50,000 and 27,000 daltons, whereas L. major promastigotes displayed a single polypeptide of 63,000 daltons. Under nonreduced conditions, the radioiodinated cell-surface component of L. major shifted to a mol.wt. of 51,000 daltons, whereas only one of the three components of L. mexicana (mol.wt., 65,000 daltons) underwent a large shift (to 59,000 daltons). The different immunochemical nature of the L. mexicana cell-surface antigens was demonstrated by using different anti-Leishmania sera. The rabbit anti-promastigote serum immunoprecipitated mainly the 50,000- and 27,000-dalton L. mexicana cell-surface polypeptides, whereas the rabbit anti-amastigote serum as well as a serum from a patient with cutaneous leishmaniasis immunoprecipitated almost exclusively the 65,000-dalton polypeptide. Immunoblot studies using a rabbit antibody against the L. major deglycosylated major surface antigen gp63 confirmed the differences in nature of the 65,000- and 50,000-dalton cell-surface antigens of L. mexicana. The results obtained are discussed in the light of the differences in antigenic cell-surface expression among Leishmania isolates and their consequences in the development of a differential diagnosis of leishmaniasis.

The phospholipid and fatty acid composition of Schistosoma mansoni and of its purified tegumental membranes.

The lipid compositions of mature male and female Schistosoma mansoni and cercariae were compared to that of the hepato-pancreas of unparasitised Biomphalaria globrata (the intermediate snail host), and of red blood cells and sera of hamsters (the mammalian host). Membranes were isolated from the tegument of mature schistosomes by spontaneous release into phosphate-buffered saline, with or without vortexing, and by removal from the parasite's surface using poly-lysine beads. The phospholipid composition of the membranes prepared by the three methods showed a typical plasma membrane-like profile, with high sphingomyelin content (approximately 20%) and cholesterol to phospholipid molar ratio (0.7-1.1). The fatty acid compositions of the resolved phospholipid classes were analysed. Although the composition was in general unremarkable, a high content of eicosaenoic acid (20:1), rarely found in mammals, was noted in whole schistosomes, cercariae, the hepato-pancreas of unparasitised Biomphalaria, and the isolated tegumental membranes. Eicosaenoic acid was also found in adults of Schistosoma japonicum. Host serum lipids from normal and parasitised hamsters contained extremely low amounts of eicosaenoic acid, indicating that this fatty acid is probably continually synthesised by the parasite, probably from preformed fatty acid precursors provided by the host.

Surface proteins and antigens of adult Schistosoma mansoni tegumental membranes detached onto poly-lysine coated beads.

Poly-lysine coated beads attached readily onto Schistosoma mansoni. On detachment, the beads removed membranes from the surface of the tegument. Analysis of the proteins of the detached membranes showed that three major proteins of 94, 73 and 62 kDa were present in contrast to a more complex range of proteins present in the phosphate-buffered saline released membranes. The membranes attached to beads were radio-iodinated and the antigens examined in immunoprecipitates by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using various antisera. In addition to the well-established 32 and 20 kDa antigens of the tegument, other major antigens of 200, 25 and 11-12 kDa were iodinated in the membranes attached to the beads. The results suggest that the major antigens studied in the tegument may not correspond to the major proteins identified. The present approach shows promise for deducing the topography of the surface antigens and proteins of schistosomes.

The recognition of Schistosoma mansoni surface antigens by antibodies from patients infected with S. mansoni and S. haematobium.

Polypeptide surface antigens of Schistosoma mansoni recognized by schistosomiasis patients have been identified and their strain and species specificity investigated. Antibodies from individuals infected with S. mansoni were used in immunoprecipitation assays of 125I-labelled schistosomulum surface antigens. All individuals surveyed from St. Lucia strongly precipitated antigens of approximately Mr 38,000 to 32,000 and 20,000. These antigens were shown by two-dimensional gel electrophoresis to be the same as those recognized by experimentally immunized mice. Although individuals showed a highly heterogeneous response against total polypeptide antigens synthesized in vitro by cell-free translation of adult S. mansoni mRNA, all individuals recognized the same surface antigens. Immunoprecipitation with sera from patients infected with S. mansoni in many different parts of Africa resulted in generally the same antigens being precipitated, although a very high molecular weight antigen(s), not strongly recognized by the St. Lucian sera was also precipitated by most of the African patient sera. One serum from Ghana precipitated the high molecular weight antigen but not the low molecular weight antigens, raising the possibility of the existence of S. mansoni strain(s) exhibiting some diversity in surface antigens. The surface of S. mansoni schistosomula was found to bind strongly antibodies from individuals infected with S. haematobium, demonstrating that most surface antigens are cross-reactive. Immunoprecipitation demonstrated, however, that of the polypeptide surface antigens only the very high molecular weight antigen was recognized by anti-S. haematobium antibodies and that the 38,000 to 32,000 and 20,000 Mr antigens were species-specific. Immunoprecipitation of the polypeptide antigens derived from purified adult surface membranes demonstrated recognition of the same 32,000, 25,000 and 20,000 Mr antigens recognized by chronically infected mice. Again these antigens were found to be species-specific.

Schistosoma mansoni: complexity of antigenic and nonantigenic surface polypeptides.

Two-dimensional gel analysis of the surface polypeptides of the schistosomula stage of Schistosoma mansoni resolved a complex pattern of approximately 20 polypeptides. The majority of these were identified as immunogenic since they were immunoprecipitated with antisera from chronically infected mice and from mice vaccinated with irradiated cercariae. However, several major surface polypeptides were not immunoprecipitated by sera from infected or immune mice and were presumed to be nonantigenic.

Schistosoma mansoni: antigen preparations which induce antibodies to schistosomula surface antigens.

To determine the easiest method of raising antibodies to antigens exposed on the surface of schistosomula of Schistosoma mansoni, several crude preparations of the parasite were used to immunize mice. Schistosomula released products, whole worm homogenate, and parasite eggs all raised antibodies which bound to the surface of live schistosomula, although the anti-egg antiserum did so less strongly. Anti-schistosomula released products antiserum recognized three schistosomula surface antigens of Mr 15,000, 20,000, and 32,000, anti-whole worm homogenate recognized 20,000, 32,000, and 38,000 Mr surface antigens, and anti-egg recognized a less than 200,000 Mr surface antigen. None of these antigens was recognized when the labeled preparation was immunoprecipitated with its homologous antiserum. When these antisera were used to immunoprecipitate cell free translation products of adult worm RNA, the antischistosomula released products and anti-whole worm homogenate recognized an 11,000 Mr doublet while the anti-egg precipitated 14,000 and 44,000 Mr antigens. Other crude preparations were used to immunize rabbits; Formalin-fixed schistosomula, denuded adult worms, and purified worm tegument all induced antibodies which recognized the 20,000, 32,000, and 38,000 Mr schistosomula surface antigens.

Schistosoma mansoni surface glycoproteins. Analysis of their expression and antigenicity.

Surface glycoproteins from newly transformed schistosomula of Schistosoma mansoni have been identified by surface radioiodination and lectin-affinity chromatography. From the glycoconjugates bound by the three lectins used, concanavalin A, peanut agglutinin and fucose-binding protein, only in the concanavalin-A-bound fractions were glycoproteins identified. Changes in concanavalin-A-binding glycoproteins were detected after transformation and early maturation of the schistosomula. Some glycoproteins disappeared (Mr 38 000, 29 000 and 25 000), some appeared independently of host molecules (Mr 19 000), others only appeared after culture in human serum (Mr 45 000). Two major glycoproteins of Mr 32 000 and 16 000 were detected on all stages examined. Within the total set of surface glycoproteins identified on 3-h schistosomula only the strong Mr-38 000-32 000 complex was found to be antigenic. Thus many major low-molecular-mass surface glycoproteins of the parasite are not recognised as antigens by immune animals. The separation of only the Mr-38 000-32 000 antigens by concanavalin A affinity chromatography indicates the feasibility of using this method in conjunction with immunoaffinity columns to purity these molecules.

Evidence that the 32, 38 and 20 kilodalton surface antigens of schistosomula and schistosomes are a family of conserved proteins.

The structures of the 38, 32 and 20 kDa surface antigens isolated from schistosomula and adult worms of Schistosoma mansoni were compared by two-dimensional peptide mapping, by immunological analysis and by one- and two-dimensional electrophoresis. Peptide mapping showed a high degree of similarity between the isolated antigens from both parasite stages. The NIMP/M47 monoclonal antibody showed cross-reactivity between the 32 and the 20 kDa antigens under denaturating and non-denaturating conditions, as demonstrated by immunoprecipitation and Western blotting. It is concluded that these antigens constitute a homologous family of surface antigens.

Changes in the surface antigen profile of Schistosoma mansoni during maturation from cercaria to adult worm.

Antigenic proteins on the surfaces of different developmental stages of Schistosoma mansoni were radio-iodinated by the Iodogen-catalysed method and identified by immunoprecipitation with a panel of antisera. The sera comprised specific immune serum from mice harbouring a chronic schistosome infection or vaccinated with gamma-irradiated cercariae; serum from rabbits immunized with adult schistosome tegumental outer membranes or a partially purified Mr 32 K glycoprotein from adult worm membranes; and a monoclonal antibody recognizing an Mr 20 K antigen on the surface of schistosomula. The Mr 38-32 K glycoproteins were the major antigens identified in surface-labelled cercariae and their probable association with the glycocalyx is discussed. Schistosomula transformed from cercariae either mechanically or by penetration of host skin in vitro, expressed a similar pattern of surface antigens to that identified for cercariae, but low molecular weight antigens of Mr 20, 17 and 15 K were also detected. The Mr 38-32 K glycoproteins, although present on newly transformed schistosomula, were progressively replaced with time, by a single dominant glycoprotein (Mr 32 K) expressing identical epitopes to those on the Mr 38-32 K complex. Moreover, the data confirm that the Mr 32 K glycoprotein persists on the tegument after in vivo maturation and is conserved, together with Mr 20 and 15 K antigens, through to the adult stage. New antigens (Mr 97 and 25 K) were also detected during in vivo maturation and were present in late-stage schistosomes recovered from infected hosts. In addition, the enzyme alkaline phosphatase is expressed on the surfaces of 3-week-old liver worms as a dominant antigen (Mr 65 K); this feature may be related to nutritional and/or physiological processes in the tegument of this metabolically active stage of development.

Schistosoma mansoni: neurotransmitters and the mobility of cercariae and schistosomules.

The concentration-dependent action of alkyl-isothiouroniums on Schistosoma mansoni cercariae, ranging from partial to total abolition of locomotor and flame cell movements, and/or suppression of virulence, is due to H1-histamine receptor inhibition. Correspondingly, H1-receptor inhibitors of widely different chemical structure, such as clemizol, diphenhydramine, brompheniramine, and promethazine, in 0.03-0.06 nM concentrations, induced an analogous cercarial immobilization reversed by addition of excess histamine. In contrast, the H2-receptor inhibitors cimetidine and metiamide did not immobilize cercariae. Histamine, acetylcholine, and serotonin, added to cercarial suspensions, showed no direct activity. Their participation in the mechanism of cercarial mobility was shown by the dose-dependent effects of antagonists, such as the serotonin antagonist methysergide and the acetylcholinesterase inhibitor physostigmine. These effects were not reversible by addition of serotonin and acetylcholine, respectively. A histamine-irreversible cercarial immobilization induced by the H-liberator 48/80 suggested that, besides H1-receptor inhibition, H-liberation and/or depletion also participated in mobility and survival. The detection of histamine in the cercaria corroborated the participation of histaminergic mechanisms. S. mansoni schistosomules collected from the mouse lung reacted to H1 antihistamines like cercariae, with a dose-dependent reduction of mobility and somatic deformation, such as vacuolization, granulation, and caecal enlargement.

Antigenicity and immunogenicity of the tegumental outer membrane of adult Schistosoma mansoni.

The tegumental membranes of adult Schistosoma mansoni have been isolated and purified and shown to function as potent immunogens; they elicit an essentially identical immune response in rabbits, rats and mice. Anti-membrane antisera harvested from these animals consistently recognized common antigens, of relative molecular weight (mol. wt) 32 000 and 20 000, on the surface of young schistosomula, 5 day old lung worms and adult worm purified membranes. An additional molecule of 25 000 mol. wt was present on the surface of lung worms and adult worm membranes and was specifically recognised by serum from chronically infected mice and by serum from rabbits inoculated with adult worm purified membranes. The concept of antigenic identity between developmental stages that parasitize the mammalian host was further substantiated by the observation that anti-membrane antiserum bound to live schistosomula, lung worms and adult parasites as measured by indirect immunofluorescence. In complement-mediated in vitro cytotoxicity assays, the sera from rabbits inoculated with either adult worm purified membranes, or the 32 000 mol. wt antigen partially purified from adult worm membranes, mediated levels of schistosomula killing as high as those obtained with sera from chronically infected mice. These rabbit antisera also promoted eosinophil adherence and killing of newly transformed schistosomula, but lung stage parasites, despite binding the anti-membrane antiserum, were refractory to both humoral and cellular cytotoxicity. The significance of antigenic identity is discussed in relation to the concept of concomitant immunity.

The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni.

Five low m.w. polypeptide antigens are expressed on the surface of freshly transformed schistosomula of Schistosoma mansoni, and were reproducibly identified by surface labeling with 125I by using IODOGEN and immunoprecipitating with immune mouse sera. These molecules have approximate m.w. of 38,000, 32,000, 20,000, 17,000, and 15,000. They correspond to antigens recognized previously by lactoperoxidase-catalyzed iodination. Analysis of the surface of developing schistosomulum demonstrated that the 38,000 and 17,000 dalton antigens were lost from the parasite surface during 48 hr of in vitro culture. This process was not dependent on the presence of host serum. The two antigens were not lost due to shedding into the culture medium but were apparently sequestered to a site where they were no longer available for surface labeling. The 32,000, 20,000, and 15,000 dalton antigens, however, remained exposed on the schistosomulum surface for up to 2 days of in vitro culture. The expression of two new antigens was also induced by culture in vitro: a doublet of approximately 45,000 daltons and an antigen of approximately 11,000 daltons. The expression of the former was dependent on the presence of serum. These results demonstrate that the development of the schistosomula surface is a complex process, with events both dependent and independent of the presence of serum. In addition, the expression of polypeptide antigens is not coordinated, and antigens are lost, retained, or appear on the schistosomulum surface during the early stages of maturation.